Hydroxychloroquine is approved for the treatment of non-cancerous illnesses such as rheumatoid arthritis and systemic lupus erythematous. Carter AE, Eban R. Prevention of postoperative deep venous thrombosis in legs by orally administered hydroxychloroquine sulphate. 2014;155(1):134–44. Cancer Res. Hydroxychloroquine reverses platelet activation induced by human IgG antiphospholipid antibodies. Arch Intern Med. Ansari D, Ansari D, Andersson R, Andren-Sandberg A. Pancreatic cancer and thromboembolic disease, 150 years after Trousseau. 2016;35(10):1078-85. 2013;210(11):2447–63. Demers M, Wong SL, Martinod K, Gallant M, Cabral JE, Wang Y, et al. Cancer Investig. All experimental animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (Protocol # 14084123). Neutrophil extracellular traps kill bacteria. Treatment of NET supernatant with DNase reversed the effects of NETs on platelet aggregation, suggesting that DNA released from neutrophils is critical for the increased aggregation. J Thromb Haemost. Removing DNA from NET supernatant using DNase I treatment prior to exposure to whole blood reversed the treatment effects of NET supernatant on platelet aggregation in human blood (a, 25.9 ± 2.2 vs. 11.35 ± 0.31, n = 4, p < 0.05). Large area scan images were captured with a Nikon A1confocal microscope (NIS Elements 4.4, Tokyo, Japan). 2012;32(8):1777–83. Nuclei were stained with Hoechst dye (bisbenzamide 1 mg/100 ml water) for 30 s. After three rinses with PBS, sections were cover slipped with Gelvatol mounting media. Hydroxychloroquine is a relatively inexpensive drug currently available for the treatment of malaria and autoimmune diseases. Thromb Res. Following standard IHC deparaffinization protocol, sections were subject to antigen retrieval using 10 mM Citric acid buffer. Data analysis was then performed using the aggrolink-8 software (ChronoLog). Importantly, treatment with CQ in PAD4 KO mice, incapable of forming NETs, had minimal effect, suggesting that CQ decreases platelet aggregation through inhibition of NETs. Thromb Haemost. Because autophagy is critical to the process of NET formation, we studied the novel use of the autophagy inhibitor chloroquine to target NET mediated hypercoagulability. Hydroxychloroquine has been shown to inhibit autophagy. While these findings point to extracellular DNA and RAGE promoting NET mediated platelet aggregation, there are many components released from NETs that may also have an impact on hypercoagulability and were not evaluated in the current analysis. Hemmers S, Teijaro JR, Arandjelovic S, Mowen KA. 2014;66(9):2532–44. Tissue factor, a transmembrane receptor typically found in subendothelial cells that binds to factor VII to initiate the extrinsic pathway when the endothelium is damaged is also released from neutrophils during NET formation [25, 26]. Front Immunol. Individuals with the following cancers are eligible if diagnosed and treated within the past three years: cervical cancer in situ, and basal cell or squamous cell carcinoma, HIV-positive individuals on combination antiretroviral therapy. Part of Pancreatic tumor specimens from resected patients with pancreatic adenocarcinoma were stained for neutrophil elastase (red) and fibrinogen (white). Furthermore, treatment of whole blood from RAGE KO mice with NET supernatant led to diminished platelet aggregation compared with WT mice (Fig. 2d). Autophagy is a catabolic pathway that permits cells to recycle intracellular macromolecules, and its inhibition reduces pancreatic cancer growth in model systems. Median PFS follow-up in this study cohort was 46.5 days (95% CI 33-61). Article  To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor. Platelet activation was assessed by analyzing expression of P-selectin (CD62P) by flow cytometry using an APC-conjugated anti-CD62P monoclonal antibody (2 μg/ml, mouse IgG1κ; eBioscience, San Diego, CA) or isotype control antibody (eBioscience) in platelet rich plasma (PRP), obtained by platelet isolation centrifugation. To investigate if DNA was the primary contributor to activating platelets in the tumor bearing mice, we treated NET supernatant with DNase I prior to mixing with whole blood ex vivo. In our recent randomized trial evaluating two months of preoperative hydroxychloroquine treatment in patients with pancreatic cancer, the VTE rate was lower in patients receiving HCQ compared to patients receiving gemcitabine/nab-paclitaxel alone. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Google ScholarÂ. Front Immunol. Binimetinib may stop the growth of tumor cells … Cells were initially plated in Hank’s Balanced Salt Solution (HBSS, Gibco, Grand Island, NY, USA), then to form NETs, HBSS was removed and cells were stimulated with 500 nM phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, MO, USA) in RPMI. Buprenex (0.1 mg/kg IP BID for 3 days) was administered for postoperative pain control. CAS  ], Progression-Free Survival [ Time Frame: Disease was evaluated radiologically at baseline and every 2 months on treatment. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis. PubMed Google Scholar. Hydroxychloroquine is an autophagy inhibitor. Neutrophils are also a source of tissue factor, as it is released during NET formation [25, 26]. Human tumor xenografts respond to combinations of hydroxychloroquine and chemotherapy (11, 12). Leshner M, Wang S, Lewis C, Zheng H, Chen XA, Santy L, et al. volume 18, Article number: 678 (2018) Nosal R, Jancinova V, Danihelova E. Chloroquine: a multipotent inhibitor of human platelets in vitro. 2014;34(9):1977–84. Rafael Pharmaceuticals, a company specializing in the field of cancer metabolism, has obtained orphan drug stats from the US Food and Drug Administration (FDA) for CPI-613 (devimistat). Extracellular DNA traps promote thrombosis. Arthritis Rheum. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. CQ treatment led to a decrease in circulating tissue factor in tumor bearing mice (d, 186.9 ± 5.6 vs. 228.2 ± 21 pg/mL, p < 0.05). Olsson AK, Cedervall J. NETosis in Cancer - platelet-neutrophil crosstalk promotes tumor-associated pathology. Platelets were gated based on their characteristic scatter properties. 2015;135(3):472–8. Data are expressed as mean ± standard deviation. Boone BA, Bahary N, Zureikat AH, Moser AJ, Normolle DP, Wu WC, et al. Although designed and powered to study the effects of HCQ on pathologic treatment response and decrease in Ca 19–9, the reduction in VTE rate neared statistical significance. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 2007;18(10):1660–5. However, our group and others have demonstrated that chloroquine prevents NET formation [13, 14]; therefore some of the antiplatelet effects of HCQ may be secondary to reduction in NET mediated DNA release which increases platelet aggregation. Inhalation or under the curve ( AUC ), ketamine ( 90 mg/kg IP BID for 3 ). The role of neutrophil extracellular trap formation is not required for immunity against influenza infection remained on.. Until the day before surgery 1R35GM119526–01 ( MDN ) on platelet activation was assessed by measuring % positive... 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